Genetic characterization of Escherichia coli and Klebsiella spp. from humans and poultry in Nigeria

The emergence of antibiotic resistance in livestock, especially food-producing animals, is of major public health importance as a result of the possibility of these bacteria entering the food chain. In this study, the genetic characteristics of antibiotic-resistant Escherichia coli and Klebsiella spp. isolates from humans and poultry in Edo state, Nigeria, were investigated. In April 2017, 45 Klebsiella spp. and 46 E. coli isolates were obtained from urine, clinical wounds, nasal and chicken faecal samples. Isolates were recovered and identified as previously described. Species identification was achieved by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry and ribosomal multilocus sequence typing. Antimicrobial susceptibility testing was carried out using the Kirby–Bauer method for 12 antibiotics. A double disc synergy test was used to screen for extended-spectrum beta-lactamse (ESBL) production. Whole genome sequencing was performed for strain characterization of the isolates. Thirteen Klebsiella spp. isolates yielded positive results by the ESBL phenotypic test and harboured ESBL genes. Of the 46 E. coli isolates, 21 human and 13 poultry isolates were resistant to at least one of the tested antibiotics. Four human E. coli isolates harboured ESBL genes and revealed positive results when applying ESBL double disc synergy tests. ESBL genes in the Klebsiella spp. and E. coli isolates include bla CTX-M-15 and bla SHV-28. Whole genome-based core gene multilocus sequence typing of the Klebsiella spp. and E. coli isolates revealed a close relatedness among the isolates. An integrated ‘One Health’ surveillance system is required to monitor transmission of antimicrobial resistance in Nigeria.


INTRODUCTION
Antimicrobial resistance (AMR) is an increasing worldwide health challenge that is regarded as a 'One Health' concern affecting human health, animal health and the environment. Antimicrobials are usually used for prophylaxis and therapeutic purposes in humans and animals. The use of antimicrobials especially in livestock for disease control and promotion of growth promotes the selection and propagation of resistant strains. These resistant strains can be transferred from the animals to humans and the environment [1]. The main mechanisms of antibiotic resistance include limiting uptake of a drug, modification of a drug target, and inactivation and active efflux of a drug [2]. These mechanisms may be intrinsically present or be acquired by mutations or transfer of resistance determinants. AMR is most commonly associated with elements that are extra-chromosomally located, including different types of mobile genetic elements such as plasmids, transposons and integrons acquired from other bacteria [3]. A large number of antimicrobials are used in poultry farming in most countries including Nigeria [4,5]. Nigeria's poultry production has grown steadily and the unrestricted use of antimicrobials is common. Resistance in Escherichia coli, Staphylococcus spp., Salmonella spp., Bacillus spp. and Klebsiella spp. has been previously reported in poultry farms [6,7]. Previous Nigerian studies OPEN ACCESS have reported resistance in isolates from poultry to some important antibiotics such as tetracycline, ciprofloxacin, sulphonamides, gentamicin and trimethoprim [4,8].
AMR in species of the family Enterobacteriaceae, especially Escherichia coli and Klebsiella pneumoniae, has spread globally causing infections that are difficult to treat [9,10]. Only a few Nigerian studies have characterized antibiotic resistance genes in poultry isolates [4,11,12]. Genetic characterization of AMR genes may give further insight into transmission mechanisms representing a key prerequisite for control of the spread of AMR [13]. The present study aims to characterize AMR E. coli and Klebsiella spp. isolates recovered from humans and poultry in Edo state Nigeria using whole genome sequencing.

Study site and sample collection
In April 2017, urine samples (130) and nasal samples (50) were obtained from healthy students of the College of Pharmacy, Igbinedion University Okada, Edo, Nigeria. The sample sizes were randomly determined. Blinding of participants was not required for the study. No inclusion or exclusion criteria were used in obtaining the samples. The samples were transported to the Department of Pharmaceutical Microbiology Laboratory immediately after collection. Isolates from clinical wounds (70) were collected from the medical microbiology laboratory of the University of Benin teaching hospital, Edo state, Nigeria. Isolates obtained were from samples of both inpatients and outpatients of the hospital. During the same period, four poultry farms situated in Okada and Benin city Edo state, Nigeria, were visited. All farms were visited once and 100 chicken faecal samples were collected and transported to the Department of Pharmaceutical Microbiology Laboratory immediately after collection. Identification of isolates was achieved using standard microbiological techniques [14]. E. coli and Klebsiella spp. were isolated and identified by inoculating samples on MacConkey agar plates (Oxoid) and incubating for 24 h at 37 °C. Distinct colonies obtained from the agar plates were subcultured to obtain pure colonies. Species identification was achieved by using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) MS (Bruker Daltonik) analysis.

Ethical considerations
Research ethics approval was not required in the hospital where isolates were obtained for the study. Only pre-identified isolates were obtained from the microbiology laboratories and used in the study. There was no contact with patients and the samples from the hospital. The institution did not require informed consent. Data regarding the isolates were obtained from clinical records and analysed anonymously. Concerning the samples obtained from the healthy students, approval from the Igbinedion university ethical committee was duly obtained for this study. The ethical document iuo/ethics/22/009 was initiated for the study. Samples were obtained from both male and female students. Data on the age and weight of the students were not obtained for the study. All participants were informed about the study's purpose and procedures. Informed consent was obtained from the students before samples were collected.

ESBL detection and antibiotic susceptibility testing
The Kirby-Bauer susceptibility testing technique [15] was carried out and results were interpreted using European Committee on Antimicrobial Susceptibility Testing criteria [16]. The isolates were tested with 12 antibiotics: meropenem, ertapenem, cefotaxime, amoxicillin/clavulanic acid, cefoxitin, cefepime, tigecycline, ciprofloxacin, amikacin, ampicillin, cefuroxime and gentamicin (Oxoid). The isolates were tested for the production of extended-spectrum beta lactamses (ESBLs) using the double disc synergy test [17]. Confirmation of ESBL production was carried out as previously described in the CLSI guidelines [18]. An ESBL producer was defined by an increase of ≥5 mm in the inhibition zone diameter for cefotaxime or ceftazidime combined with clavulanic acid against the inhibition zone diameter of either cefotaxime or ceftazidime without clavulanic acid [18]. Based on a random selection criterion, some antibiotic-resistant and ESBL-positive isolates were selected for characterization by whole genome sequencing (WGS).

Whole genome sequencing
WGS was carried out as previously described [1] for 16 resistant/multidrug-resistant E. coli and Klebsiella spp. isolates respectively based on a random selection criterion. Briefly, genomic DNA (gDNA) was extracted from the isolates using MagAttract HMW DNA extraction kit (Qiagen). Fragment libraries of the bacterial genomes were prepared using the Illumina Nextera XT DNA library preparation kit (Illumina). Library preparation of the genomes was followed by paired-end sequencing using a read length of 2×300 bp on an Illumina Miseq instrument (Miseq v3.0; Illumina). Assembly of raw reads (FASTQ files) was carried out using Velvet version 1.1.04 [19]. Ribosomal multilocus sequence typing (rMLST; https://pubmlst.org/species-id) was used to confirm the identities of the isolates. The ResFinder 2.1 web server tool (http://www.genomicepidemiology.org) [20] and the Comprehensive Antibiotic Resistance Database-Resistance Gene Identifier (CARD-RGI) [21] were used to identify plasmids and AMR genes in assembled genomes. The VirulenceFinder 2.0 web server tool (http://www.genomicepidemiology.org) [22] and the VFDB [23] were used to identify virulence factors in assembled genomes of E. coli and Klebsiella spp. isolates respectively. O-and H-types in the E. coli isolates were identified using SerotypeFinder 2.0 (http://www.genomicepidemiology.org) [24]. Mobile genetic elements and their relationship to AMR genes and virulence factors in assembled genomes were predicted using the MobileElementFinder (v1.0.2) (http://www.genomicepidemiology.org) [25]. For phylogenetic analysis, the MLST (multi-locus sequence typing) [26] and the K. pneumoniae core genome MLST (cgMLST) (https://www.cgmlst.org/ncs/schema/2187931/) were determined using SeqSphere+software v6.0.0 (Ridom). For strain comparison of Klebsiella spp., minimum spanning trees (MSTs) were calculated based on the cgMLST scheme comprising 2358 core genes using SeqSphere+ software v6.0.0 (Ridom). A CT distance of 15 alleles was used to identify related isolates. The MLST and cgMLST scheme comprising 2513 core genes was used for E. coli strain comparison using SeqSphere+ version 6.0.0 (Ridom). Related E.coli isolates were identified with a CT distance of 10 alleles. 'Good core genome targets' was defined based on the criteria previously described in detail in Ruppitsch et al. [27]. Plasmids on the draft genomes of the resistant E. coli and Klebsiella spp. isolates were detected and classified using the PlasmidFinder 1.3 webtool (https://cge.food.dtu.dk/services/PlasmidFinder/) [28]. Replication of experiments was not required in the study.

Antibiogram and phenotypic resistance pattern of the isolates
Of the 46 E. coli isolates, 21 human (two nasal, 12 urine, seven wound) and 13 poultry isolates were antibiotic resistant in the Kirby-Bauer susceptibility testing technique. Four human (three wound, one nasal) E. coli isolates showed positive results in the ESBL double disc synergy tests. The ESBL E. coli isolates were all resistant to cefepime, cefuroxime, cefotaxime and gentamicin. Only one ESBL E. coli was resistant to ciprofloxacin. Twelve human (nine wound, two urine, one nasal) and one poultry Klebsiella spp. isolates showed positive results when applying ESBL double disc synergy tests. All ESBL Klebsiella spp. isolates were resistant to amoxicillin clavulanic acid, cefuroxime and cefotaxime. Eleven ESBL Klebsiella spp. isolates were cefepime-resistant. Ten and seven ESBL Klebsiella spp. isolates were also resistant to ciprofloxacin and gentamicin respectively (Figs 1 and 2).

AMR genes of the recovered resistant isolates
ESBL genes detected among the isolates included bla CTX-M-15 and bla SHV-28 . Multiple resistance determinants were detected on the draft genome sequence of the 12 human and four poultry E. coli isolates analysed (Table S1, available in the online version of this article). The resistance determinants included β-lactamase genes, aminoglycoside modifying enzymes, fosfomycin resistance determinants, qnr genes, sulphonamide resistance genes, tetracycline resistance genes, trimethoprim resistance genes and phenicol resistance genes. Other resistance determinants, which included efflux regulatory systems modulating antibiotic efflux, antibiotic target alteration and elfamycin resistance, were also detected in the antibiotic-resistant E. coli isolates. Eleven plasmid incompatibility groups were identified among the 16 resistant isolates with IncF family types being predominant. WGS also confirmed the presence of other β-lactamase genes, aminoglycoside-modifying enzymes, fosfomycin resistance determinants, qnr genes and plasmid encoded efflux pump, sulphonamide resistance genes, trimethoprim resistance genes, phenicol resistance genes among the resistant Klebsiella spp. isolates (Table S1). Thirteen plasmid incompatibility groups were identified among the resistant Klebsiella spp. isolates with IncF family types also being predominant.

Virulence factors of the recovered resistant isolates
Known virulence determinants involved in adherence, biofilm formation, capsule synthesis regulation, immune evasion, secretion system, serum resistance, siderophore expression (enterobactin, yersiniabactin, aerobactin and salmochelin) and efflux pump expression were detected among the Klebsiella spp. isolates (Table S1). The chromosomal gene fim, mrk for adherence and biofilm formation, the immune evasion factor cpsA which determines the polysaccharide capsule (K antigen) type, capsule synthesis regulation (rcs) serum resistance factor (OmpA) which determines the 'O-antigen' lipopolysaccharide serotype and efflux pump expression (acrAB) were identified among the Klebsiella spp. isolates. Other factors identified among the Klebsiella spp. isolates include factors the determine siderophores (iron carriers) [aerobactin (iutA), salmochelin (iroE, iroN) and enterobactin (ent, fep)] and type VI secretion system loci genes (Table S1).
For the E. coli isolates, all 16 isolates were predicted to be of four different serogroup O types by SerotypeFinder 2.0, and seven different H types were detected. Among the isolates, three (19 %) were identified as serotype O1:H25. Serotypes O8:H9, O7:H4, O8:H17 and O16:H5 were detected in four different E. coli isolates. Nine (56 %) E. coli isolates had only the serogroup H type by SerotypeFinder 2.0, which was predominantly H9 in six (38 %) E. coli isolates. MLST analysis showed that the six E. coli isolates with serogroup H9 belonged to sequence type (ST)155, three isolates with serogroup O1:H25 belonged to ST1722 and two isolates with ST10 were predicted to have serogroup H27. Six different FimH types (23,24,32,41,54,153) were identified among all the E. coli isolates except one in which it was not detected. Different virulence factors belonging to the four main virulence classes of E. coli pathotypes, namely colonization, fitness, toxins, and effectors, were identified among the E. coli isolates (Table S1). Some virulence genes were commonly detected among the E. coli isolates, which include hlyE, fimH, gad, nlpl, csgA and terC (Table  S1). Other virulence factors detected include chuA, coding for an outer membrane haemin receptor, yfcV (a fimbriae adhesin), iha, air, tia and lpfA adhesins, fyuA expressing a siderophore receptor, senB which codes for an enterotoxin, eilA, a Salmonella HilA homologue, hra, a heat-resistant agglutinin gene, irp2, encoding a non-ribosomal peptide synthetase, papC, encoding outer membrane usher P fimbriae, iss, an increased serum survival protein, cnf1, a cytotoxic necrotizing factor, kpsE a capsule polysaccharide export inner-membrane protein, and traT, an outer membrane complement resistance protein (Table S1).

Characterization of mobile genetic elements
MobileElementFinder (http://www.genomicepidemiology.org) predicted five types of mobile genetic elements (MGEs) among the resistant isolates, which include miniature inverted repeats, insertion sequences (ISs), an integrative conjugative element, unit transposons and composite transposons of which the majority were ISs. Some of the predicted ISs among the resistant isolates include ISEcl1, ISEc9, ISEc31, IS6100, IS26, ISKpn19 and ISKpn26. No integrative conjugative element ICEEoED1a-1 or miniature inverted repeat MITEEc1 was detected in E. coli and Klebsiella spp. isolates respectively. The majority of the resistant isolates had mobile elements in association with AMR and virulence genes. Seven E. coli isolates had mobile elements with no association with AMR or virulence genes (Table S1).

Genetic comparison of Klebsiella spp. and E. coli isolates
Whole genome-based cgMLST phylogenetic analysis of Klebsiella spp. isolates was performed and a MST was calculated (Fig. 4). Distance calculation between the 16 samples revealed a maximum allelic distance between samples of 2867 across the MST. Based on the defined complex threshold (CT) of 15 allelic differences, two different clusters were obtained containing five isolates from clinical wounds. The isolates were closely related with a maximum allelic difference of four. Cluster 1 had three ST2728 isolates with similar plasmid incompatibility group IncFIB(K), IncFII(K). Cluster 2 had two ST16 isolates with similar plasmid incompatibility group Col440II, IncFIB(K), IncFII(K). Phylogenetic analysis of E. coli isolates was also performed and an MST was calculated based on cgMLST (Fig. 3). Distance calculation between the 16 samples revealed a maximum allelic distance between samples of 2347 across the MST. Based on the defined CT of 10 allelic differences, three different clusters were obtained. The isolates in cluster 1 (Fig. 3) included four isolates from urine of healthy humans and two isolates from healthy poultry animals. The isolates (ST155 and a common plasmid incompatibility group p0111, IncQ1, IncFII) were closely related with a maximum allelic difference of three. The farms where the poultry samples were obtained are in the same location as the healthy individuals. Cluster 2 consists of an ST1722 isolate from a healthy individual (nasal sample) and two ST1722 isolates from clinical wounds that were closely related with an allelic difference of one. The isolates had a common plasmid incompatibility group IncFIA, IncFIB. The location where the isolates from clinical wounds were obtained is not in close proximity to the location where nasal samples of the healthy individuals were collected. Cluster 3 includes two ST10 isolates with similar plasmid incompatibility group IncFII from clinical wounds.

DISCUSSION
AMR is of global health concern and is increasingly frustrating therapeutic efforts against infectious diseases. This has been linked to the misuse and/or overuse of antimicrobials in humans and animals [9,11,29]. The present study determined the frequency of antimicrobial-resistant E. coli and Klebsiella spp. isolates recovered from humans and poultry in selected areas of Edo state, Nigeria, and provides the genetic characterization of the isolates. Humans and the poultry environment have been previously reported to be reservoirs for antimicrobial-resistant bacteria [9,11,[29][30][31]. The frequency of resistant isolates from humans was higher compared to isolates from poultry. Results from this study show that Klebsiella spp. and E. coli isolates especially from poultry were resistant to antibiotics commonly used in human and veterinary medicine. Although bans on the non-therapeutic use of antimicrobials exist in multiple countries including Nigeria, the coordinated surveillance and monitoring of the use of antimicrobials and their resistance in Nigeria is still limited, which has resulted in an increase of antimicrobial-resistant pathogens.
One limitation of this study was the failure to document the use of specific antimicrobials on the farms where samples were collected. Previous studies from developed countries have demonstrated the clonal spread of bacteria between human and animal reservoirs while only a few studies are available from sub-Saharan Africa [30]. Due to limited discriminatory power, classical MLST is not adequate to reaveal the clonal spread of bacteria between human and animal reservoirs. Results from this study suggest clonal transmission of E. coli between the two reservoirs (healthy humans and poultry). This correlates with a previous publication that reported potential clonal transmission between the two reservoirs (humans and poultry) in a rural Ghanaian town [30]. Another recent African study, which also correlates with results from the present study, showed a complex relationship between multi-resistant E. coli isolates in poultry and humans indicating that multi-resistance control must involve a One-Health approach and a multi-sectoral collaboration [32]. In our study, the isolation of closely related E. coli ST1722 isolates from humans that were not in close proximity to each other furthermore indicates clonal spread of resistant bacteria in the human community. The lower level of hygiene in developing countries compared to developed countries facilitates inter-host transmission of bacteria. Previous reports have indicated that contaminated poultry products are a significant source for acquisition of resistant bacteria due to hand contamination and cross-contamination during meal preparation [8,33,34]. In our study, no ESBL-producing E. coli could be isolated from poultry. This contrasts with previous reports of ESBL-producing E. coli detected from poultry [30,35,36]. In our study, bla CTX-M-15 was predominantly present in the resistant isolates obtained from humans. This correlates with a previous study from Nigeria revealing that bla CTX-M-15 constitutes the most frequent ESBL type detected in clinical human Enterobacteriaceae isolates [9]. In our study, the frequency of detection of ESBL bla CTX-M-15 in bacteria from faecal poultry samples was considerably lower than in human isolates.
E. coli ST155, which had a high frequency in human and poultry isolates, has been previously reported as responsible for transmission of ESBL genes and plasmid-mediated spread of antibiotic resistance from animals to humans [37,38]. It has acquired adaptive traits that increase pathogenicity, colonization and spread in different kinds of niches [39,40]. A recent Nigerian study reported E. coli ST155 as one of the most common STs detected among ESBL E coli strains from human, chickens, and chicken market environments [41]. The findings demonstrated that the co-colonization of antimicrobial-resistant E. coli from a shared source is also possible, which correlates with results obtained from this study. K. pneumoniae ST307 is an important clone that has been identified in diverse locations worldwide and is associated with the ESBL gene bla CTX-M-15 [42,43]. K. pneumoniae ST307 has been involved in local hospital outbreaks in Africa, the Americas, Asia and Europe [44] with limited reports on non-human sources in Africa [45]. The detection of K. pneumoniae ST307 harbouring bla CTX-M-15 in poultry is of public health importance as it confirms that not only humans could serve as reservoir for this clone. To the best of our knowledge, our results present the first identification of K. pneumoniae ST307 bla CTX-M-15 in poultry in Nigeria. Virulence genes were detected in all the Klebsiella spp. and E. coli isolates, which suggests poultry may be a reservoir for genes encoding virulence in addition to human sources. K. pneumoniae has four important virulence factors, adhesive fimbriae (including type 1 and type 3 fimbriae), capsule, LPS and siderophores, that contribute to the pathogenicity of K. pneumoniae isolates. Siderophores were the most significant virulence genes detected among the Klebsiella spp. isolates. A previous report [46] showed that acquiring the virulence factor yersiniabactin is usually the initial step in accumulating more potent siderophores to make isolates more invasive. Yersiniabactin genes were found among eight human K. pneumoniae isolates and and one poultry isolate. All isolates with yersiniabactin genes were resistant/multidrug-resistant and ESBL-positive, making their infections persistent and antibiotic-resistant and possibly contributing to their dominance over the other pathogroups. The entB gene is a siderophore-associated gene of K. pneumoniae. Iron is required for bacterial survival. K. pneumoniae usually acquires iron via the secretion of siderophores, which have a higher affinity for iron than host transport proteins [47]. In this study, the entB gene was detected in 100 % of the human and poultry Klebsiella spp. isolates which agrees with other reports [47,48]. FimH and mrkD are other important virulent factors detected among the Klebsiella spp. isolates essential in pili formation which allows for adherence of the pathogen and for formation of biofilms on biotic and abiotic surfaces. In this present study, the rates of detection of the fimH and mrkD genes among the human and poultry Klebsiella spp. isolates were 100 and 81.3 %, respectively, which is consistent with the results obtained in previous studies [47][48][49]. The presence of varying virulence markers in the E. coli isolates suggests different pathogroups present among the E. coli isolates. A previous classification of Uropathogenic E. coli (UPEC) considers the presence of at least two or more of the following genes: chuA, fyuA (coding for ferric yersinia uptake yersiniabactin receptor), vat and yfcV (adhesin) [50]. Significantly, four E. coli isolates in which three were ESBL ST1722 with predicted serotype O1:H25 had the virulence factor fyuA detected in association with yfcV and chuA, confirming them as possible UPEC pathotypes. Previously, Johnson et al. [51] classified bacterial strains as possible Extraintestinal pathogenic E. coli (ExPEC) isolates when possessing two or more of the following virulence determinants: papAH, and/or papC (P fimbriae), sfa-focDE (S and F1C fimbriae), afa-draBC (Dr-binding adhesins), iutA (aerobacting siderophore system) and kpsM II (group 2 capsules). None of these genes were in the same E. coli isolate in this study. However, some genes, such as papC or kpsM II, in association with other virulence markers were recovered in the study. These isolates could be considered commensals, coding for ExPEC-associated virulence genes. Another frequent virulence marker detected among the E. coli isolates is lpfA. Previous studies detected lpfA in enteropathogenic E. coli (EPEC), cattle shiga toxin-producing E. coli (STEC), extra-intestinal pathogenic E. coli and commensal E. coli [52,53]. This gene was detected in human as well as poultry E. coli isolates in this study. In the present study, 100 % of the human and poultry E. coli isolates possessed multi-virulence-associated genes. These results agreed with previous studies that reported the majority of E. coli isolates to have at least three virulence-associated genes [54,55]. The results of this study indicated that the examined E. coli and Klebsiella spp. strains in both human and poultry isolates possess a characteristic set of virulence factors and some of these virulent factors associated with AMR genes may be of public health concern as a result of their potential to cause human infections [29,56]. Using the MobileElementFinder web tool, a variety in the number and combination of MGEs was identified simplifying the detection and characterization of MGEs and their relationship to AMR and virulence genes for this study. ISs and other transposable elements have previously been reported to be associated with the mobilization of antibiotic-resistant determinants and the transmission of pathogenic characteristics [57]. The detection of MGEs in most of the resistant isolates associated with antimicrobial-resistant and virulence genes is of public health concern as they might possibly facilitate the transmission of these genes among the isolates. A good understanding of the genetic diversity of an organism is necessary to understand its transmission dynamics and to predict its source and potential for transmission from animals to humans [58].The importance of WGS as a vital genomic surveillance tool to detect and characterize the genetic basis of Nigerian human and poultry Klebsiella spp. and E. coli isolates has been demonstrated in this study This study describes the detection of ESBL-producing/antimicrobial-resistant bacteria with varying virulence profiles in humans and also poultry samples from Edo state in Nigeria. The results show that poultry farms or meat products might be an important source for ESBL-producing/antimicrobial-resistant pathogenic bacteria. The importance of genomic approaches in the surveillance of AMR for improving the prevention and control of infection in Nigerian hospitals cannot be underestimated. The benefit of public health genomic surveillance of pathogens and its potential for outbreak detection is of great importance in low-and middle-income countries. An integrated 'One Health' surveillance system involving collaboration among scientists, health institutions and public health authorities is required to monitor transmission of AMR in Nigeria.

Funding information
This work was supported financially by the Austrian Agency for Health and Food Safety (AGES), Vienna, Austria.

Peer review history
We believe that the revised manuscript is now suitable for publication in ACCESS MICROBIOLOGY.

Dr. Christiana Jesumirhewe
On behalf of all authors.

Reviewers' comments and responses to custom questions:
1. Methodological rigour, reproducibility and availability of underlying data Clear methodology with reproducible techniques. Whole genome Illumina sequencing was used for further bioinformatic analysis, which was appropriate.
Line 73: is information of the bed capacity needed?
Line 73 has been removed from the manuscript

Presentation of results
Tables highlight the results accurately, however maybe another way to present this would be easier to follow (Table 1 and Table  2)? Perhaps via graphs using other statistical programs (R etc). More bioinformatic analyses outputs can be highlighted in figure format vs. written in the text as this makes it easier to follow and provides evidence. Table 1 and 2 has been re-presented as Figure 1 and 2

Literature analysis or discussion
Indication of key results and limitations to own research were layed out well. Limitations of other papers and advantages of the current study were mentioned but not discussed well/enough.

Any other relevant comments
Line 58: Refer to Nigerian studies, please show which ones and highlight these in your references.

Line 58 Nigerian studies highlighted and shown in references
Line 113: By paired end sequencing

Line 113 corrected to By paired end sequencing
Line 133: Missing full stop at end of paragraph.

Full stop included at end of paragraph Line 133
Reviewer 2 Comments to Author:

Methodological rigour, reproducibility and availability of underlying data
Some parts need further information to allow replication studies

Presentation of results
A very General Description of the isolates. The manuscript would benefit from a more detailed description of the isolates and extension of the in silico analysis especially for mobile genetic element, transmissible resistance and virulence factors as well as an assessment of the potential impact of the isolates for human (virulence factors) Manuscript has been revised extending analysis for mobile genetic element, transmissible resistance and virulence factors as well as an assessment of the potential impact of the isolates for human (virulence factors) 3. How the style and organization of the paper communicates and represents key findings Style is ok, but the key findings can be presented in more detail.

Any other relevant comments
The manuscript would benefit of a general revision and additional in silico analysis using the prevailing data. This reviewer suggest to conduct this as the manuscript lacks depth.

Manuscript has been revised and is more detailed now Editor comments:
This study would be a valuable contribution to the existing literature. The reviewers have highlighted minor concerns with the work presented. Please ensure that you address their comments. Reviewer 2 recommended the following: ...extension of the in silico analysis especially for mobile genetic element, transmissible resistance and virulence factors as well as an assessment of the potential impact of the isolates for human (virulence factors)... If you choose not to perform further analysis, please ensure all data and descriptions are available to allow others to perform further analysis on your data should they wish, and describe how your data might be used by yourselves or others within your field in the future.

Comments have been addressed as required by both reviewers
Editorial Office requirements: 1 Please move the information regarding your funding to it's own 'Funding information' section, as required by the platform. factors) 3. How the style and organization of the paper communicates and represents key findings Style is ok, but the key findings can be presented in more detail. 4. Literature analysis or discussion Ok 5. Any other relevant comments The manuscript would benefit of a general revision and additional in silico analysis using the prevailing data. This reviewer suggest to conduct this as the manuscript lacks depth.

Please rate the manuscript for methodological rigour Satisfactory
Please rate the quality of the presentation and structure of the manuscript Good To what extent are the conclusions supported by the data? Partially support Comments: Overall clearly written paper. The importance of the issue of AMR is highlighted in every part of the paper with clear emphasis of combatting this issue through awareness and a "one health" approach. 1. Methodological rigour, reproducibility and availability of underlying data Clear methodology with reproducible techniques. Whole genome Illumina sequencing was used for further bioinformatic analysis, which was appropriate. Line 73: is information of the bed capacity needed? 2. Presentation of results Tables highlight the results accurately, however maybe another way to present this would be easier to follow (Table 1 and  Table 2)? Perhaps via graphs using other statistical programs (R etc). More bioinformatic analyses outputs can be highlighted in figure format vs. written in the text as this makes it easier to follow and provides evidence. 3. How the style and organization of the paper communicates and represents key findings Clear organisation and style of writing. Methodological order with clear results subtitles and text, which is also reflected in the discussion. Each key finding was stated and evidence of other studies highlighting this matter were shown. 4. Literature analysis or discussion Indication of key results and limitations to own research were layed out well. Limitations of other papers and advantages of the current study were mentioned but not discussed well/ enough. 5. Any other relevant comments Line 58: Refer to Nigerian studies, please show which ones and highlight these in your references. Line 113: By paired end sequencing* Line 133: Missing full stop at end of paragraph.

Please rate the manuscript for methodological rigour Good
Please rate the quality of the presentation and structure of the manuscript Good To what extent are the conclusions supported by the data? Strongly support Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices? No